Multiple discrete regions at 8q24 were recently shown to contain alleles that predispose to many cancers including prostate, breast, and colon. These regions are far from any annotated gene and their biological activities have been unknown. Here we profiled a 5-megabase chromatin segment encompassing all the risk regions for RNA expression, histone modifications, and locations occupied by RNA polymerase II and androgen receptor (AR). This led to the identification of several transcriptional enhancers, which were verified using reporter assays. Two enhancers in one risk region were occupied by AR and responded to androgen treatment; one contained a single nucleotide polymorphism (rs11986220) that resides within a FoxA1 binding site, with the prostate cancer risk allele facilitating both stronger FoxA1 binding and stronger androgen responsiveness. The study reported here exemplifies an approach that may be applied to any risk-associated allele in non-protein coding regions as it emerges from genome-wide association studies to better understand the genetic predisposition of complex diseases.

Genetically tractable fruit flies have been used for decades to study seizure disorders. However, there is a paucity of data specifically correlating fly and human seizure phenotypes. We have previously shown that mutation of orthologous PRICKLE genes from flies to humans produce seizures. This study aimed to determine whether the prickle-mediated seizure phenotypes in flies closely parallel the epilepsy syndrome found in PRICKLE patients.

In past years, much attention has focused on the gene networks that regulate early developmental processes, but less attention has been paid to how multiple networks and processes are temporally coordinated. Recently the discovery of the transcriptional activator Zelda (Zld), which binds to CAGGTAG and related sequences present in the enhancers of many early-activated genes in Drosophila, hinted at a mechanism for how batteries of genes could be simultaneously activated. Here we use genome-wide binding and expression assays to identify Zld target genes in the early embryo with the goal of unraveling the gene circuitry regulated by Zld. We found that Zld binds to genes involved in early developmental processes such as cellularization, sex determination, neurogenesis, and pattern formation. In the absence of Zld, many target genes failed to be activated, while others, particularly the patterning genes, exhibited delayed transcriptional activation, some of which also showed weak and/or sporadic expression. These effects disrupted the normal sequence of patterning-gene interactions and resulted in highly altered spatial expression patterns, demonstrating the significance of a timing mechanism in early development. In addition, we observed prevalent overlap between Zld-bound regions and genomic "hotspot" regions, which are bound by many developmental transcription factors, especially the patterning factors. This, along with the finding that the most over-represented motif in hotspots, CAGGTA, is the Zld binding site, implicates Zld in promoting hotspot formation. We propose that Zld promotes timely and robust transcriptional activation of early-gene networks so that developmental events are coordinated and cell fates are established properly in the cellular blastoderm embryo.

Proper gene regulation is critical for both neuronal development and maintenance as the brain matures. We previously demonstrated that Akirin2, an essential nuclear protein that interacts with transcription factors and chromatin remodeling complexes, is required for the embryonic formation of the cerebral cortex. Here we show that Akirin2 plays a mechanistically distinct role in maintaining healthy neurons during cortical maturation. Restricting Akirin2 loss to excitatory cortical neurons resulted in progressive neurodegeneration via necroptosis and severe cortical atrophy with age. Comparing transcriptomes from Akirin2-null postnatal neurons and cortical progenitors revealed that targets of the tumor suppressor p53, a regulator of both proliferation and cell death encoded by Trp53, were consistently upregulated. Reduction of Trp53 rescued neurodegeneration in Akirin2-null neurons. These data: (1) implicate Akirin2 as a critical neuronal maintenance protein, (2) identify p53 pathways as mediators of Akirin2 functions, and (3) suggest Akirin2 dysfunction may be relevant to neurodegenerative diseases.

We used paired-end next generation sequencing (NGS) to characterize the classic isoform-specific pk pk1 and pk sple1 mutations of the prickle gene in Drosophila melanogaster . Here we provide evidence that these previously reported null mutations are caused by either a tirant transposon insertion into the 5' UTR of pk pk1 or a premature stop codon in the second exon of pk sple1 . Additional likely benign missense mutations were identified in both mutant isoforms.

Polycomb-group (PcG) and Trithorax-group proteins together form a maintenance machinery that is responsible for stable heritable states of gene activity. While the best-studied target genes are the Hox genes of the Antennapedia and Bithorax complexes, a large number of key developmental genes are also Polycomb (Pc) targets, indicating a widespread role for this maintenance machinery in cell fate determination. We have studied the linkage between the binding of PcG proteins and the developmental regulation of gene expression using whole-genome mapping to identify sites bound by the PcG proteins, Pc and Pleiohomeotic (Pho), in the Drosophila embryo and in a more restricted tissue, the imaginal discs of the third thoracic segment. Our data provide support for the idea that Pho is a general component of the maintenance machinery, since the majority of Pc targets are also associated with Pho binding. We find, in general, considerable developmental stability of Pc and Pho binding at target genes and observe that Pc/Pho binding can be associated with both expressed and inactive genes. In particular, at the Hox complexes, both active and inactive genes have significant Pc and Pho binding. However, in comparison to inactive genes, the active Hox genes show reduced and altered binding profiles. During development, Pc target genes are not simply constantly associated with Pc/Pho binding, and we identify sets of genes with clear differential binding between embryo and imaginal disc. Using existing datasets, we show that for specific fate-determining genes of the haemocyte lineage, the active state is characterised by lack of Pc binding. Overall, our analysis suggests a dynamic relationship between Pc/Pho binding and gene transcription. Pc/Pho binding does not preclude transcription, but levels of Pc/Pho binding change during development, and loss of Pc/Pho binding can be associated with both stable gene activity and inactivity.

Vesicoureteral reflux (VUR) is a complex, heritable disorder. Genome-wide linkage analyses of families affected by VUR have revealed multiple genomic loci linked to VUR. These loci normally harbor a number of genes whose biologically functional variant is yet to be identified. DNA copy number variations (CNVs) have not been extensively studied at high resolution in VUR patients. In this study, we performed array comparative genomic hybridization (aCGH) on a cohort of patients with a history of both VUR and urinary tract infection (UTI) with the objective of identifying genetic variations responsible for VUR and/or UTI susceptibility. UTI/VUR-associated CNVs were identified by aCGH results from the 192 Randomized Intervention for Children With Vesicoureteral Reflux (RIVUR) patients compared to 683 controls. Rare, large CNVs that are likely pathogenic and lead to VUR development were identified using stringent analysis criteria. Because UTI is a common affliction with multiple risk factors, we utilized standard analysis to identify potential disease-modifying CNVs that can contribute to UTI risk. Gene ontology analysis identified that CNVs in innate immunity and development genes were enriched in RIVUR patients. CNVs affecting innate immune genes may contribute to UTI susceptibility in VUR patients and may provide the first step in assisting clinical medicine in determining adverse outcome risk in children with VUR.

Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in prickle mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of fat facets a USP9X orthologue. USP9X mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a de novo missense mutation and another a novel coding mutation. Both USP9X variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress prickle-mediated seizure activity, and that USP9X variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum.

We report the development of OikoBase (http://oikoarrays.biology.uiowa.edu/Oiko/), a tiling array-based genome browser resource for Oikopleura dioica, a metazoan belonging to the urochordates, the closest extant group to vertebrates. OikoBase facilitates retrieval and mining of a variety of useful genomics information. First, it includes a genome browser which interrogates 1260 genomic sequence scaffolds and features gene, transcript and CDS annotation tracks. Second, we annotated gene models with gene ontology (GO) terms and InterPro domains which are directly accessible in the browser with links to their entries in the GO (http://www.geneontology.org/) and InterPro (http://www.ebi.ac.uk/interpro/) databases, and we provide transcript and peptide links for sequence downloads. Third, we introduce the transcriptomics of a comprehensive set of developmental stages of O. dioica at high resolution and provide downloadable gene expression data for all developmental stages. Fourth, we incorporate a BLAST tool to identify homologs of genes and proteins. Finally, we include a tutorial that describes how to use OikoBase as well as a link to detailed methods, explaining the data generation and analysis pipeline. OikoBase will provide a valuable resource for research in chordate development, genome evolution and plasticity and the molecular ecology of this important marine planktonic organism.

Paraoxonases (PON) are a family of proteins (PON1, 2 and 3) with multiple enzymatic activities. PON1 interferes with homoserine lactone-mediated quorum sensing in bacteria and with reactive oxygen species (ROS) in humans and mice. PON1 gene mutations have been linked to multiple traits, including aging, and diseases of the cardiovascular, nervous and gastrointestinal system. The overlapping enzymatic activities in the PON family members and high linkage disequilibrium rates within their polymorphisms confound animal and human studies of PON1 function. In contrast, arthropods such as Drosophila melanogaster have no PON homologs, resulting in an ideal model to study interactions between PON genotype and host phenotypes. We hypothesized that expression of PON1 in D. melanogaster would alter ROS. We found that PON1 alters expression of multiple oxidative stress genes and decreases superoxide anion levels in normal and germ-free D. melanogaster. We also found differences in the composition of the gut microbiota, with a remarkable increase in levels of Lactobacillus plantarum and associated changes in expression of antimicrobial and cuticle-related genes. PON1 expression directly decreased superoxide anion levels and altered bacterial colonization of the gut and its gene expression profile, highlighting the complex nature of the interaction between host genotype and gut microbiota. We speculate that the interaction between some genotypes and human diseases may be mediated by the presence of certain gut bacteria that can induce specific immune responses in the gut and other host tissues.

As opposed to syndromic CNVs caused by single genes, extensive phenotypic heterogeneity in variably-expressive CNVs complicates disease gene discovery and functional evaluation. Here, we propose a complex interaction model for pathogenicity of the autism-associated 16p11.2 deletion, where CNV genes interact with each other in conserved pathways to modulate expression of the phenotype. Using multiple quantitative methods in Drosophila RNAi lines, we identify a range of neurodevelopmental phenotypes for knockdown of individual 16p11.2 homologs in different tissues. We test 565 pairwise knockdowns in the developing eye, and identify 24 interactions between pairs of 16p11.2 homologs and 46 interactions between 16p11.2 homologs and neurodevelopmental genes that suppress or enhance cell proliferation phenotypes compared to one-hit knockdowns. These interactions within cell proliferation pathways are also enriched in a human brain-specific network, providing translational relevance in humans. Our study indicates a role for pervasive genetic interactions within CNVs towards cellular and developmental phenotypes.

Mutations in the gene encoding transcription factor TFAP2A result in pigmentation anomalies in model organisms and premature hair graying in humans. However, the pleiotropic functions of TFAP2A and its redundantly-acting paralogs have made the precise contribution of TFAP2-type activity to melanocyte differentiation unclear. Defining this contribution may help to explain why TFAP2A expression is reduced in advanced-stage melanoma compared to benign nevi. To identify genes with TFAP2A-dependent expression in melanocytes, we profile zebrafish tissue and mouse melanocytes deficient in Tfap2a, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including Dct, Mc1r, Mlph, and Pmel. We then conduct TFAP2A ChIP-seq in mouse and human melanocytes and find that a much larger subset of pigmentation genes is associated with active regulatory elements bound by TFAP2A. These elements are also frequently bound by MITF, which is considered the "master regulator" of melanocyte development. For example, the promoter of TRPM1 is bound by both TFAP2A and MITF, and we show that the activity of a minimal TRPM1 promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of Trpm1 is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs Tfap2a and Tfap2b are both expressed in mouse melanocytes, and we show that mouse embryos with Wnt1-Cre-mediated deletion of Tfap2a and Tfap2b in the neural crest almost completely lack melanocytes but retain neural crest-derived sensory ganglia. These results suggest that TFAP2 paralogs, like MITF, are also necessary for induction of the melanocyte lineage. Finally, we observe a genetic interaction between tfap2a and mitfa in zebrafish, but find that artificially elevating expression of tfap2a does not increase levels of melanin in mitfa hypomorphic or loss-of-function mutants. Collectively, these results show that TFAP2 paralogs, operating alongside lineage-specific transcription factors such as MITF, directly regulate effectors of terminal differentiation in melanocytes. In addition, they suggest that TFAP2A activity, like MITF activity, has the potential to modulate the phenotype of melanoma cells.

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution.

Completion of chromosome condensation is required before segregation during the mitotic cell cycle to ensure the transmission of genetic material with high fidelity in a timely fashion. In many eukaryotes this condensation is regulated by phosphorylation of histone H3 on Ser 10 (H3S10). This phosphorylation normally begins in the late-replicating pericentric heterochromatin and then spreads to the earlier replicating euchromatin. Here, we show that these phases of condensation are genetically separable in that the absence of Drosophila Myb causes cells to arrest with H3S10 phosphorylation of heterochromatin but not euchromatin. In addition, we used mosaic analysis to demonstrate that although the Myb protein can be removed in a single cell cycle, the failure of chromosome condensation occurs only after many cell divisions in the absence of Myb protein. The Myb protein is normally located in euchromatic but not heterochromatic regions of the nucleus, suggesting that Myb may be essential for a modification of euchromatin that is required for the efficient spread of chromosome condensation.

An inherited variant on chromosome 8q24, rs6983267, is significantly associated with cancer pathogenesis. We present evidence that the region harboring this variant is a transcriptional enhancer, that the alleles of rs6983267 differentially bind transcription factor 7-like 2 (TCF7L2) and that the risk region physically interacts with the MYC proto-oncogene. These data provide strong support for a biological mechanism underlying this non-protein-coding risk variant.

Chromatin immunoprecipitation combined with genome tile path microarrays or deep sequencing can be used to study genome-wide epigenetic profiles and the transcription factor binding repertoire. Although well studied in a variety of cell lines, these genome-wide profiles have so far been little explored in vertebrate embryos.

Drosophila Myb (Dm-Myb) encodes a protein that plays a key role in regulation of mitotic phase genes. Here, we further refine its role in the context of a developing tissue as a potentiator of gene expression required for proper RNA polymerase II (RNA Pol II) function and efficient H3K4 methylation at promoters. In contrast to its role in gene activation, Myb is also required for repression of many genes, although no specific mechanism for this role has been proposed. We now reveal a critical role for Myb in contributing to insulator function, in part by promoting binding of insulator proteins BEAF-32 and CP190 and stabilizing H3K27me3 Polycomb-group (PcG) domains. In the absence of Myb, H3K27me3 is markedly reduced throughout the genome, leading to H3K4me3 spreading and gene derepression. Finally, Myb is enriched at boundaries that demarcate chromatin environments, including chromatin loop anchors. These results reveal functions of Myb that extend beyond transcriptional regulation.

Predator-induced defences are a prominent example of phenotypic plasticity found from single-celled organisms to vertebrates. The water flea Daphnia pulex is a very convenient ecological genomic model for studying predator-induced defences as it exhibits substantial morphological changes under predation risk. Most importantly, however, genetically identical clones can be transcriptionally profiled under both control and predation risk conditions and be compared due to the availability of the sequenced reference genome. Earlier gene expression analyses of candidate genes as well as a tiled genomic microarray expression experiment have provided insights into some genes involved in predator-induced phenotypic plasticity. Here we performed the first RNA-Seq analysis to identify genes that were differentially expressed in defended vs. undefended D. pulex specimens in order to explore the genetic mechanisms underlying predator-induced defences at a qualitatively novel level.

Animals have developed extensive mechanisms of response to xenobiotic chemical attacks. Although recent genome surveys have suggested a broad conservation of the chemical defensome across metazoans, global gene expression responses to xenobiotics have not been well investigated in most invertebrates. Here, we performed genome survey for key defensome genes in Oikopleura dioica genome, and explored genome-wide gene expression using high density tiling arrays with over 2 million probes, in response to two model xenobiotic chemicals - the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) the pharmaceutical compound Clofibrate (Clo).